Femtosecond study of light-induced fluorescence increase of the dark chromoprotein asFP595

Tanja A. Schüttrigkeit, Till von Feilitzsch, Christian K. Kompa, Konstantin A. Lukyanov, Alexander P. Savitsky, Alexander A. Voityuk, Maria E. Michel-Beyerle

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31 Citations (Scopus)


Femtosecond time-resolved spectroscopy is applied to study the mechanism of the light-induced increase of fluorescence quantum yield of the initially non-fluorescent (dark) chromoprotein asFP595. Spectroscopic and kinetic characteristics of this unique fluorescence "kindling" phenomenon are: (i) the small Stokes shift of the dark chromophore consistent with either the zwitterion or the anion; (ii) the singlet excited state of the dark chromophore decaying predominantly with a time constant of ∼320 fs corresponding to a fluorescence quantum yield ΦFl {less-than or slanted equal to} 10-4. Since ground state recovery occurs on the same time scale, this radiationless channel is assigned to internal conversion; (iii) the formation of the fluorescent species depending on the sequential absorption of two photons with a delay significantly exceeding the excitation pulse duration of 150 fs; (iv) the fluorescent species showing a red-shift of ∼20 nm in absorption and emission, and an excited state lifetime of 2.2 ns. The ultrafast internal conversion of the excited dark state is attributed to the proximity of the S0 and S1 potential energy surfaces favored by the non-planarity of the chromophore as revealed in recent X-ray structures. Competing with internal conversion two different transformations of the chromophore structure are suggested which may be identified in a future X-ray structural analysis of the the photoconverted fluorescent state. The predominant kindling mechanism may be either (i) trans-cis isomerization or (ii) proton transfer between an excited zwitterion and the protein cleft. For mechanism (ii) the large dipole moment change of about 11 D upon S0-S1 excitation of the chromophore would be crucial in order to initiate protein relaxation and deprotonation of a zwitterion. Both mechanisms are assumed to lead to a metastable planar structure responsible for the long-lived fluorescence of the chromophore "kindled" at high light intensities.

Original languageEnglish
Pages (from-to)149-160
Number of pages12
JournalChemical Physics
Issue number2-3
Publication statusPublished - 21 Apr 2006
Externally publishedYes


  • asFP595
  • Femtosecond spectroscopy
  • Fluorescence kindling mechanism
  • Red fluorescent protein


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